*Target (protein/gene name):Protein phosphatase 2C, putative/Tc00.1047053506315.100 *NCBI Gene # or RefSeq#: Tc00.1047053506315.100 *Protein ID (NP or XP #) or Wolbachia#: Q4D795 *Organism (including strain):Tyrypanosoma cruzi Etiologic Risk Group (see link below): Risk Group 2 Background/Disease Information (sort of like the Intro to your Mini Research Write up):Trypanosoma cruzi is a parasitic organism that causes the disease known as Chagas disease. This parasite begins it's life in an animal reservoir with the triatomine bug acting as the vector that transmits the eukaryote to the human blood stream. Once in the human body, these parasites go through a period of reproduction which causes the cells host cells to burst and release the new parasites. Symptoms of Chagas disease include myocardial symptoms that involve conduction abnormalities in the heart. Since the heart muscle operates through electrical signals, these abnormalities can cause sudden death in patients. As a result of these effects, patients who are diagnosed with Chagas disease tend to die due to heart failure. While some treatments include nifurtimox and benznidazole, there is no current long-term treatment for this disease. Essentiality of this protein: This is a protein phosphatase meaning that it is needed in the dephosphorylation of molecules in Trypanosoma cruzi. Dephosphorylation works in tandem with phosphorylation in order for cells to be able to use ATP to function. One instance where this type of protein is needed is in signal cascades. The dephosphorylation/phosphorylation of a different protein could lead to a cascade of other signals in the cells that might possibly trigger the synthesis of other essential proteins. Complex of proteins?: This protein is not found necessarily in a "complex" but rather interacts extensively with other proteins during it's operations. Druggable Target: The target is druggable. *EC#: 3.1.3.16 Link to BRENDA EC# page: http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.1.3.16 Enzyme Assay information: http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/calcineurin.pdf http://www.sigmaaldrich.com/catalog/product/sigma/t1503?lang=en®ion=US
For a full list of reagents needed, refer to pages five and six of the PDF linked above. All products are available through Sigma Aldrich.
Structure Available (PDB or Homology model) Current Inhibitors: There are currently no well-known inhibitors of this specific protein although there are drug therapies available to treat the disease. It should be noted that no long-term treatment has been discovered however. Expression Information (has it been expressed in bacterial cells): This protein has been previously expressed in the bacterial cells of E. coli. Purification Method: Refer to the assay methods as well as the procedures used in previous experiments involving this protein. Image of protein (PyMol with features delineated and shown separately):
(It should be noted that this is not a PyMol representation of the protein in question because of the fact that there is no PDB available. Instead, this is the image of the protein that has the best homology match and might be viable for use as a model for the phosphatase to be studied.) *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): 10 20 30 40 50 60 MPSSSKKNRG KNAVHSNNNN NHSVNHRGGP VKSKKQEESD EFEKVLADLS RATEVAGTKR
370 380 390 400 410 ACVLSKKQQG KSNLVICEDI CRECLAPPAE GGGRSSRAEG TDNMTIMIVD LK *length of your protein in Amino Acids: 412 amino acids Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 40175.0 kDa Molar Extinction coefficient of your protein at 280 nm wavelength: 20315 TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html): This website does not recognize the given sequence for this particular protein. *CDS Gene Sequence (paste as text only):
ATGCCGAGCAGCAGCAAAAAGAACAGGGGTAAGAATGCCGTCCACAGCAATAATAATAAT AATCACTCTGTGAACCATAGAGGCGGCCCCGTAAAGAGCAAAAAGCAGGAGGAGAGTGAT GAGTTTGAGAAGGTTCTGGCGGACCTCAGCAGGGCTACAGAGGTTGCAGGCACGAAGAGG GCCAAGATGCTAGAGCGTGATGCTAAGATGCGGGTGGAGCGGGTGGAGAGGTATAAGAAC GTCATTGCCGGGAGGGCAAAGGAGGCCGAAATGGAGTTTGACATGATGTTGCGCCGTAAA CAGCAGAAGCAGCAGTTCCAGGATCTCCTTCAACAGCTGCTAGCCGCCCAACGTGTGCTC CTGGACGCCCCCAATACCGACTTCCAAGGCGAAACAGTGACGGAAAATCCTCATTTTGAC GTAGCGGTAGGGGAGATGCAGGGCTGGCGTGTCAACATGGAGGATGAGCACCTGGTAGAT GTCAAATTCCCGAACGATGAGCCCGATAGCAAGGAGGGACTCTTCTGCGTTTTTGATGGC CACTCGGGGAAGGGGTGTGCCAAGAAGTGTCGGGAGTTGATTTCCAAGACAGCGAGGAAA TACTGGACGCGTGTGACGGGGGAAAGCGCGAGTTCCACGGTGGACTTTGAGAAAGTGTAC CTGGAGGTGGACGGCATTTTAGAGAAAGAGTTGACGGATGGATCAGGTTGCACAGCTGTC ACGGTACACGTCACATCCGATGTTATTACCTGCGCCTCTGTCGGAGACTCCCGTGCGGTG CTTTGCCGCAACGGCGCTGCCTTTTACCTTTCGTACGACCACAAACCAGAGAATGCGTTG GAGCGCGAAAGGATCGAGTCTGCAGGAGGTTCTGTCTCGGAGAACCGCGTAAATGGCCAA CTTGCGATGAGCCGAGCGATGGGTGATTTCATCTACAAGAATCAAAAGGATCGTGACCCG AGGGAGCAGCATGTCATTGCCGTGCCTGATGTTATCAGCACCCCGCGGGAGGCCGGTGAC ACATTTGTGGTGTTGGCCTGCGATGGTATTTTTGACGTCCTTGGAAACGATGAACTGGTC GCTTGTGTGCTTTCCAAGAAGCAACAGGGCAAAAGTAACTTGGTGATATGTGAGGATATT TGTCGTGAGTGTTTGGCGCCACCGGCGGAGGGCGGTGGTCGCAGCTCACGCGCCGAGGGC ACAGACAACATGACCATCATGATCGTCGATCTTAAGTAA *GC% Content for gene: 53.83% *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
1 AGCAGCAGCA AAAAGAACAG GGGTAAGAAT GCCGTCCACA GCAATAATAA
51 TAATAATCAC TCTGTGAACC ATAGAGGCGG CCCCGTAAAG AGCAAAAAGC
101 AGGAGGAGAG TGATGAGTTT GAGAAGGTTC TGGCGGACCT CAGCAGGGCT
151 ACAGAGGTTG CAGGCACGAA GAGGGCCAAG ATGCTAGAGC GTGATGCTAA
201 GATGCGGGTG GAGCGGGTGG AGAGGTATAA GAACGTCATT GCCGGGAGGG
251 CAAAGGAGGC CGAAATGGAG TTTGACATGA TGTTGCGCCG TAAACAGCAG
301 AAGCAGCAGT TCCAGGATCT CCTTCAACAG CTGCTAGCCG CCCAACGTGT
351 GCTCCTGGAC GCCCCCAATA CCGACTTCCA AGGCGAAACA GTGACGGAAA
401 ATCCTCATTT TGACGTAGCG GTAGGGGAGA TGCAGGGCTG GCGTGTCAAC
451 ATGGAGGATG AGCACCTGGT AGATGTCAAA TTCCCGAACG ATGAGCCCGA
501 TAGCAAGGAG GGACTCTTCT GCGTTTTTGA TGGCCACTCG GGGAAGGGGT
551 GTGCCAAGAA GTGTCGGGAG TTGATTTCCA AGACAGCGAG GAAATACTGG
601 ACGCGTGTGA CGGGGGAAAG CGCGAGTTCC ACGGTGGACT TTGAGAAAGT
651 GTACCTGGAG GTGGACGGCA TTTTAGAGAA AGAGTTGACG GATGGATCAG
701 GTTGCACAGC TGTCACGGTA CACGTCACAT CCGATGTTAT TACCTGCGCC
751 TCTGTCGGAG ACTCCCGTGC GGTGCTTTGC CGCAACGGCG CTGCCTTTTA
801 CCTTTCGTAC GACCACAAAC CAGAGAATGC GTTGGAGCGC GAAAGGATCG
851 AGTCTGCAGG AGGTTCTGTC TCGGAGAACC GCGTAAATGG CCAACTTGCG
901 ATGAGCCGAG CGATGGGTGA TTTCATCTAC AAGAATCAAA AGGATCGTGA
951 CCCGAGGGAG CAGCATGTCA TTGCCGTGCC TGATGTTATC AGCACCCCGC
1001 GGGAGGCCGG TGACACATTT GTGGTGTTGG CCTGCGATGG TATTTTTGAC
1051 GTCCTTGGAA ACGATGAACT GGTCGCTTGT GTGCTTTCCA AGAAGCAACA
1101 GGGCAAAAGT AACTTGGTGA TATGTGAGGA TATTTGTCGT GAGTGTTTGG
1151 CGCCACCGGC GGAGGGCGGT GGTCGCAGCT CACGCGCCGA GGGCACAGAC
1201 AACATGACCA TCATGATCGT CGATCTT *GC% Content for gene (codon optimized): 53.95%
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
*NCBI Gene # or RefSeq#: Tc00.1047053506315.100
*Protein ID (NP or XP #) or Wolbachia#: Q4D795
*Organism (including strain): Tyrypanosoma cruzi
Etiologic Risk Group (see link below): Risk Group 2
Background/Disease Information (sort of like the Intro to your Mini Research Write up): Trypanosoma cruzi is a parasitic organism that causes the disease known as Chagas disease. This parasite begins it's life in an animal reservoir with the triatomine bug acting as the vector that transmits the eukaryote to the human blood stream. Once in the human body, these parasites go through a period of reproduction which causes the cells host cells to burst and release the new parasites. Symptoms of Chagas disease include myocardial symptoms that involve conduction abnormalities in the heart. Since the heart muscle operates through electrical signals, these abnormalities can cause sudden death in patients. As a result of these effects, patients who are diagnosed with Chagas disease tend to die due to heart failure. While some treatments include nifurtimox and benznidazole, there is no current long-term treatment for this disease.
Essentiality of this protein: This is a protein phosphatase meaning that it is needed in the dephosphorylation of molecules in Trypanosoma cruzi. Dephosphorylation works in tandem with phosphorylation in order for cells to be able to use ATP to function. One instance where this type of protein is needed is in signal cascades. The dephosphorylation/phosphorylation of a different protein could lead to a cascade of other signals in the cells that might possibly trigger the synthesis of other essential proteins.
Complex of proteins?: This protein is not found necessarily in a "complex" but rather interacts extensively with other proteins during it's operations.
Druggable Target: The target is druggable.
*EC#: 3.1.3.16
Link to BRENDA EC# page:
http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.1.3.16
Enzyme Assay information:
http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/calcineurin.pdf
http://www.sigmaaldrich.com/catalog/product/sigma/t1503?lang=en®ion=US
For a full list of reagents needed, refer to pages five and six of the PDF linked above. All products are available through Sigma Aldrich.
Structure Available (PDB or Homology model)
Current Inhibitors: There are currently no well-known inhibitors of this specific protein although there are drug therapies available to treat the disease. It should be noted that no long-term treatment has been discovered however.
Expression Information (has it been expressed in bacterial cells): This protein has been previously expressed in the bacterial cells of E. coli.
Purification Method: Refer to the assay methods as well as the procedures used in previous experiments involving this protein.
Image of protein (PyMol with features delineated and shown separately):
(It should be noted that this is not a PyMol representation of the protein in question because of the fact that there is no PDB available. Instead, this is the image of the protein that has the best homology match and might be viable for use as a model for the phosphatase to be studied.)
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
10 20 30 40 50 60
MPSSSKKNRG KNAVHSNNNN NHSVNHRGGP VKSKKQEESD EFEKVLADLS RATEVAGTKR
70 80 90 100 110 120
AKMLERDAKM RVERVERYKN VIAGRAKEAE MEFDMMLRRK QQKQQFQDLL QQLLAAQRVL
130 140 150 160 170 180
LDAPNTDFQG ETVTENPHFD VAVGEMQGWR VNMEDEHLVD VKFPNDEPDS KEGLFCVFDG
190 200 210 220 230 240
HSGKGCAKKC RELISKTARK YWTRVTGESA SSTVDFEKVY LEVDGILEKE LTDGSGCTAV
250 260 270 280 290 300
TVHVTSDVIT CASVGDSRAV LCRNGAAFYL SYDHKPENAL ERERIESAGG SVSENRVNGQ
310 320 330 340 350 360
LAMSRAMGDF IYKNQKDRDP REQHVIAVPD VISTPREAGD TFVVLACDGI FDVLGNDELV
370 380 390 400 410
ACVLSKKQQG KSNLVICEDI CRECLAPPAE GGGRSSRAEG TDNMTIMIVD LK
*length of your protein in Amino Acids: 412 amino acids
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 40175.0 kDa
Molar Extinction coefficient of your protein at 280 nm wavelength: 20315
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html): This website does not recognize the given sequence for this particular protein.
*CDS Gene Sequence (paste as text only):
ATGCCGAGCAGCAGCAAAAAGAACAGGGGTAAGAATGCCGTCCACAGCAATAATAATAAT AATCACTCTGTGAACCATAGAGGCGGCCCCGTAAAGAGCAAAAAGCAGGAGGAGAGTGAT GAGTTTGAGAAGGTTCTGGCGGACCTCAGCAGGGCTACAGAGGTTGCAGGCACGAAGAGG GCCAAGATGCTAGAGCGTGATGCTAAGATGCGGGTGGAGCGGGTGGAGAGGTATAAGAAC GTCATTGCCGGGAGGGCAAAGGAGGCCGAAATGGAGTTTGACATGATGTTGCGCCGTAAA CAGCAGAAGCAGCAGTTCCAGGATCTCCTTCAACAGCTGCTAGCCGCCCAACGTGTGCTC CTGGACGCCCCCAATACCGACTTCCAAGGCGAAACAGTGACGGAAAATCCTCATTTTGAC GTAGCGGTAGGGGAGATGCAGGGCTGGCGTGTCAACATGGAGGATGAGCACCTGGTAGAT GTCAAATTCCCGAACGATGAGCCCGATAGCAAGGAGGGACTCTTCTGCGTTTTTGATGGC CACTCGGGGAAGGGGTGTGCCAAGAAGTGTCGGGAGTTGATTTCCAAGACAGCGAGGAAA TACTGGACGCGTGTGACGGGGGAAAGCGCGAGTTCCACGGTGGACTTTGAGAAAGTGTAC CTGGAGGTGGACGGCATTTTAGAGAAAGAGTTGACGGATGGATCAGGTTGCACAGCTGTC ACGGTACACGTCACATCCGATGTTATTACCTGCGCCTCTGTCGGAGACTCCCGTGCGGTG CTTTGCCGCAACGGCGCTGCCTTTTACCTTTCGTACGACCACAAACCAGAGAATGCGTTG GAGCGCGAAAGGATCGAGTCTGCAGGAGGTTCTGTCTCGGAGAACCGCGTAAATGGCCAA CTTGCGATGAGCCGAGCGATGGGTGATTTCATCTACAAGAATCAAAAGGATCGTGACCCG AGGGAGCAGCATGTCATTGCCGTGCCTGATGTTATCAGCACCCCGCGGGAGGCCGGTGAC ACATTTGTGGTGTTGGCCTGCGATGGTATTTTTGACGTCCTTGGAAACGATGAACTGGTC GCTTGTGTGCTTTCCAAGAAGCAACAGGGCAAAAGTAACTTGGTGATATGTGAGGATATT TGTCGTGAGTGTTTGGCGCCACCGGCGGAGGGCGGTGGTCGCAGCTCACGCGCCGAGGGC ACAGACAACATGACCATCATGATCGTCGATCTTAAGTAA
*GC% Content for gene: 53.83%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
1 AGCAGCAGCA AAAAGAACAG GGGTAAGAAT GCCGTCCACA GCAATAATAA
51 TAATAATCAC TCTGTGAACC ATAGAGGCGG CCCCGTAAAG AGCAAAAAGC
101 AGGAGGAGAG TGATGAGTTT GAGAAGGTTC TGGCGGACCT CAGCAGGGCT
151 ACAGAGGTTG CAGGCACGAA GAGGGCCAAG ATGCTAGAGC GTGATGCTAA
201 GATGCGGGTG GAGCGGGTGG AGAGGTATAA GAACGTCATT GCCGGGAGGG
251 CAAAGGAGGC CGAAATGGAG TTTGACATGA TGTTGCGCCG TAAACAGCAG
301 AAGCAGCAGT TCCAGGATCT CCTTCAACAG CTGCTAGCCG CCCAACGTGT
351 GCTCCTGGAC GCCCCCAATA CCGACTTCCA AGGCGAAACA GTGACGGAAA
401 ATCCTCATTT TGACGTAGCG GTAGGGGAGA TGCAGGGCTG GCGTGTCAAC
451 ATGGAGGATG AGCACCTGGT AGATGTCAAA TTCCCGAACG ATGAGCCCGA
501 TAGCAAGGAG GGACTCTTCT GCGTTTTTGA TGGCCACTCG GGGAAGGGGT
551 GTGCCAAGAA GTGTCGGGAG TTGATTTCCA AGACAGCGAG GAAATACTGG
601 ACGCGTGTGA CGGGGGAAAG CGCGAGTTCC ACGGTGGACT TTGAGAAAGT
651 GTACCTGGAG GTGGACGGCA TTTTAGAGAA AGAGTTGACG GATGGATCAG
701 GTTGCACAGC TGTCACGGTA CACGTCACAT CCGATGTTAT TACCTGCGCC
751 TCTGTCGGAG ACTCCCGTGC GGTGCTTTGC CGCAACGGCG CTGCCTTTTA
801 CCTTTCGTAC GACCACAAAC CAGAGAATGC GTTGGAGCGC GAAAGGATCG
851 AGTCTGCAGG AGGTTCTGTC TCGGAGAACC GCGTAAATGG CCAACTTGCG
901 ATGAGCCGAG CGATGGGTGA TTTCATCTAC AAGAATCAAA AGGATCGTGA
951 CCCGAGGGAG CAGCATGTCA TTGCCGTGCC TGATGTTATC AGCACCCCGC
1001 GGGAGGCCGG TGACACATTT GTGGTGTTGG CCTGCGATGG TATTTTTGAC
1051 GTCCTTGGAA ACGATGAACT GGTCGCTTGT GTGCTTTCCA AGAAGCAACA
1101 GGGCAAAAGT AACTTGGTGA TATGTGAGGA TATTTGTCGT GAGTGTTTGG
1151 CGCCACCGGC GGAGGGCGGT GGTCGCAGCT CACGCGCCGA GGGCACAGAC
1201 AACATGACCA TCATGATCGT CGATCTT
*GC% Content for gene (codon optimized): 53.95%
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):